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Biomedical subjects

R H Michell

Publications and source records attributed to R H Michell.

At least 181 records · Page 10Linked to original sources
PubMed1975

Enhanced synthesis de novo of phosphatidylinositol in lymphocytes treated with cationic amphiphilic drugs.

A variety of amphiphilic cations caused very large increases in the rates of incorporation of Pi and glycerol into phosphatidylinositol in pig mesenteric small lymphocytes. This synthesis de novo of phosphatidylinositol led to a doubling of the phosphatidylinositol concentration in the cells within 3.5 h. The increase in synthesis of phosphatidylinositol labelled with [3H]- or [14C]-glycerol was matched by an approximately equivalent decrease in incorporation of glycerol into phosphatidylcholine, phosphatidylethanolamine and triacylglycerol. Amphilic cations which produced these effects included, in order of decreasing effectiveness, trifluoperazine (half-maximal effect at about 70 mum) greater than chlorpromazine approximately promethazine approximately imipramine greater than cinchocaine greater than amethocaine approximately cetyltrimethylammonium greater than fenfluramine greater than amphetamine greater than 2-phenethylamine greater than cocaine approximately procaine; the most effective compounds were those with the largest and most hydrophobic non-polar substituents. The response to cations was not changed by varying the extracellular Ca2+ concentration in the range 10 nm-1mm. The active amphiphilic cations interacted with anionic phospholipids causing aggregation of aqueous dispersions and/or changes in chromatographic behaviour. These results indicate that amphiphilic cations redirect glycerolipid synthesis de novo, probably owing to inhibition of phosphatidate phosphohydrolase, so that phosphatidylinositol synthesis is increased at the expense of other glycerolipids.

Amphetamine↗
PubMed1975

The relationship of calcium to receptor-controlled stimulation of phosphatidylinositol turnover. Effects of acetylcholine, adrenaline, calcium ions, cinchocaine and a bivalent cation ionophore on rat parotid-gland fragments.

The possibility that Ca2+ ions are involved in the control of the increased phosphatidylinositol turnover which is provoked by alpha-adrenergic or muscarinic cholinergic stimulation of rat parotid-gland fragments has been investigated. Both types of stimulation provoked phosphatidylinositol breakdown, which was detected either chemically or radiochemically, and provoked a compensatory synthesis of the lipid, detected as an increased rate of incorporation of 32Pi into phosphatidylinositol. Acetylcholine had little effect on the incorporation of labelled glycerol, whereas adrenaline stimulated it significantly, but to a much lower extent than 32P incorporation: this suggests that the response to acetylcholine was entirely accounted for by renewal of the phosphorylinositol head-group of the lipid, but that some synthesis de novo was involved in the response to adrenaline. The responses to both types of stimulation, whether measured as phosphatidylinositol breakdown or as phosphatidylinositol labelling, occurred equally well in incubation media containing 2.5 mm-Ca2+ or 0.2 mm-EGTA [ethanedioxybis(ethylamine)-tetra-acetic acid]. Incubation with a bivalent cation ionophore (A23187) led to a small and more variable increase in phosphatidylinositol labelling with 32Pi, which occurred whether or not Ca2+ was available in the extracellular medium: this was not accompanied by significant phosphatidylinositol breakdown. Cinchocaine, a local anaesthetic, produced parallel increases in the incorporation of Pi and glycerol into phosphatidylinositol. This is compatible with its known ability to inhibit phosphatidate phosphohydrolase (EC 3.1.3.4) and increase phosphatidylinositol synthesis de novo in other cells. These results indicate that the phosphatidylinositol turnover evoked by alpha-adrenergic or muscarinic cholinergic stimuli in rat parotid gland probably does not depend on an influx of Ca2+ into the cells in response to stimulation. This is in marked contrast with the K+ efflux from this tissue, which is controlled by the same receptors, but is strictly dependent on the presence of extracellular Ca2+. The Ca2+-independence of stimulated phosphatidylinositol metabolism may mean that it is controlled through a mode of receptor function different from that which controls other cell responses. Alternatively, it can be interpreted as indicating that stimulated phosphatidylinositol breakdown is intimately involved in the mechanisms of action of alpha-adrenergic and muscarinic cholinergic receptor systems.

Acetylcholine↗
PubMed1975

Basolateral plasma membranes of intestinal epithelial cells. Identification by lactoperoxidase-catalysed iodination and isolation after density perturbation with digitonin.

Lactoperoxidase-catalysed iodination was used to label intestinal epithelial cell sheets with 125I. The iodination was carried out under conditions that allowed little penetration of lactoperoxidase into the cells and membrane-bound 125I therefore provided an effective marker for following plasma-membrane fragments through subcellular-fractionation procedures. 2. After homogenization and isopycnic zonal centrifugation through sucrose gradients two peaks of membrane-bound 125I were detected. One coincided with brush border enzymes such as alkaline phosphatase, disaccharidases and L-leucine B-naphthylamidase, whereas the other was coincident with the major peak of (Na++K+)-stimulated ATPase (adenosine triphosphatase), which has been thought to be concentrated in the basolateral plasma membranes of these cells. Neither peak of 125I reflected the distribution of any marker for an intracellular organelle. 3. A larger proportion of the (Na++K+)-stimulated ATPase, and thus of the basolateral plasma-membrane material, was found in a crude 'mitochondrial' fraction. It was not readiily separated from mitochondria by conventional techniques of subcellular fractionation. 4. Treatment of the 'mitochondrial' fraction with digitonin increased the density of basolateral plasma membrane but had little effect on mitochondrial density. A purified preparation of digitonin-loaded basolateral plasma membranes was isolated at a density of 1.20-1.22 by isopycnic centrifugation. 5. The enzymic composition of this preparation of basolateral plasma membranes is compared with previous preparations isolated from intestinal mucosal 'scrape' materials and from isolated cells.

Adenosine Triphosphatases↗
PubMed1974

Enhanced phosphatidylinositol labelling in rat parotid fragments exposed to alpha-adrenergic stimulation.

1. Adrenergic agonists provoke a marked increase in labelling of phosphatidylinositol in fragments of rat parotid gland. 2. Adrenaline and phenylephrine (an adrenergic alpha-agonist) are effective stimulants, but isoprenaline (an adrenergic beta-agonist) is relatively ineffective. 3. The response evoked by phenylephrine or adrenaline is prevented by prior incubation of the tissue with phenoxybenzamine (an alpha-receptor blocking agent), but not by prior incubation with pindolol (a beta-receptor blocking agent). 4. Adrenergic stimulation of phosphatidylinositol metabolism in parotid gland is therefore mediated through alpha-receptors, in common with the adrenaline-induced K(+) efflux. It is not linked to enzyme secretion, which is triggered by stimulation of beta-receptors. 5. It is suggested that the stimulation of phospholipid metabolism that occurs in several other tissues in the presence of adrenaline or noradrenaline may also involve alpha-receptors.

Animals↗
PubMed1974

Breakdown of phosphatidylinositol provoked by muscarinic cholinergic stimulation of rat parotid-gland fragments.

When rat parotid fragments that had been labelled with (32)P in vivo were exposed to high concentrations of acetylcholine, radioactivity was lost from phosphatidylinositol but not from other phospholipids. Simultaneously the concentration of phosphatidylinositol in the tissue decreased. If previously unlabelled tissue was incubated with (32)P(i) an increase in incorporation of radioactivity into phosphatidylinositol was observed during this decrease in concentration. The effects of acetylcholine were blocked by atropine, but not by tubocurarine. The response to acetylcholine was rapid, with up to one-third of the tissue's phosphatidylinositol disappearing within 5min. Similar effects were evoked by stimulation with methacholine and by high concentrations of tetramethylammonium ion; these responses were also atropine-sensitive and tubocurarine-insensitive. It is concluded that the event in inositol lipid metabolism that is affected by acetylcholine stimulation is removal of the phosphorylinositol group from the molecule; this is mediated through muscarinic cholinergic receptors. This is followed by a compensatory increase in the rate of synthesis of phosphatidylinositol, which has been described in detail in the past. These observations are compared with those of previous workers and are discussed in relation to the existing hypotheses relating to the significance of stimulus-provoked phosphatidylinositol turnover.

Acetylcholine↗
PubMed1974

Phosphatidylinositol cleavage catalysed by the soluble fraction from lymphocytes. Activity at pH5.5 and pH7.0.

Phosphatidylinositol breakdown by subcellular preparations of small lymphocytes from pig mesenteric lymph nodes was investigated. Activity was higher than in preparations from the tissues studied previously; it was recovered largely in the soluble fraction, which showed pH optima at both 5.4-5.6 and 7.0-7.3. As in other tissues, phosphatidylinositol cleavage produced 1,2-diacylglycerol and a mixture of myo-inositol 1:2-cyclic phosphate and myo-inositol 1-phosphate. It was stimulated by addition of CaCl(2) and, less effectively, by MgCl(2). On sucrose-density-gradient ultracentrifugation at pH7.0 two peaks of activity were observed (approx. sedimentation coefficients 8S and 10S); the activity profiles on the gradients were similar when assayed at pH7.0 and 5.5. Activity at pH7.0 (and 0.4mm-CaCl(2)) was decreased by agents, such as salts and lipophilic cations, which tend to neutralize the negative charge of phosphatidylinositol; at pH5.5 these agents slightly stimulated activity. It is suggested that the same enzyme(s) may be responsible for activity at both pH optima and that previous workers may have underestimated the pH7.0 activity because of the inhibitory influence of cations under the usual assay conditions.

Animals↗
PubMed1974

Phosphatidylinositol cleavage in lymphocytes. Requirement for calcium ions at a low concentration and effects of other cations.

The soluble activity in lymphocytes which converts phosphatidylinositol into 1,2-diacylglycerol and inositol phosphates requires Ca(2+) ions. At pH7 maximum activity occurs at [Ca(2+)](free) approximately 0.7mum whereas at pH5.5 the equivalent value is approx. 50mum. At [Ca(2+)](free) approximately 1mum, a concentration similar to common intracellular values, essentially all activity is confined to the peak of activity at pH7.0. Previous reports of requirements for larger amounts of Ca(2+) may reflect the fact that the Ca(2+)-buffering capacity of phosphatidylinositol means that high substrate concentrations can effectively decrease [Ca(2+)](free). Cations which displace Ca(2+) from association with phosphatidylinositol can, at low [Ca(2+)](free), enhance enzyme activity. Phosphatidylinositol breakdown in intact cells might be controlled, at least in part, by changes in intracellular [Ca(2+)](free).

Animals↗
PubMed1973

A membrane-bound activity catalysing phosphatidylinositol breakdown to 1,2-diacylglycerol, D-myoinositol 1:2-cyclic phosphate an D-myoinositol 1-phosphate. Properties and subcellular distribution in rat cerebral cortex.

1. Breakdown of phosphatidylinositol was studied in homogenates and subcellular fractions of rat cerebral cortex by using both membrane-bound and externally added [(32)P]phosphatidylinositol as substrate. 2. In the presence of deoxycholate breakdown followed first-order kinetics at low substrate concentrations ([unk]1mm) and zero-order kinetics at higher concentrations (6-9mm). 3. Maximum breakdown by cerebral-cortex homogenates was approximately 0.5mumol/h per mg of protein and occurred at pH7.0 in the presence of 8mm-phosphatidylinositol, 2mm-CaCl(2) and 2mg of deoxycholate/ml. Activity was abolished by 1mm-ethanedioxybis(ethylamine)tetra-acetate. 4. The products of phosphatidylinositol breakdown were 1,2-diacylglycerol and a mixture of d-myoinositol 1:2-cyclic phosphate (55%) and d-myoinositol 1-phosphate (45%). The two phosphate esters appeared to be produced together and in constant proportions. 5. Some 51% of the activity was particle-bound, with the highest activities in small nerve endings, microsomal material and two synaptic membrane fractions (fractions Mic(20), Mic(100), M(1) 1.0 and M(1) 0.9 respectively), all of which were also rich in acetylcholinesterase and which have been shown to be rich in other surface-membrane enzymes. Much of the particle-bound activity therefore appears to be present in cerebral-cortex plasma membranes. 6. The results are discussed in relation to previously described soluble activities that catalyse the same reaction, and to a possible role of the membrane-bound enzyme in enhanced phosphatidylinositol turnover in externally stimulated cells.

Acetylcholinesterase↗
PubMed1972

Stimulation by acetylcholine of phosphatidylinositol labelling. Subcellular distribution in rat cerebral-cortex slices.

1. Rat cerebral-cortex slices were incubated with (32)P(i), acetylcholine and eserine for periods of 10min and 2h. The specific radioactivity of phosphatidylinositol was elevated during these treatments by 36 and 106% respectively. 2. The specific radioactivities of the phosphatidylinositol in different cell structures were determined after subcellular fractionation. They were highest in the nuclear, microsomal and synaptic-vesicle fractions and lowest in myelin, both in the controls and in the acetylcholine-treated slices. 3. The stimulated labelling of phosphatidylinositol was relatively evenly distributed: no subcellular fraction showed a stimulation markedly higher than that in the homogenate. 4. Studies of the distributions and activities of marker enzymes indicated that the subcellular fractionation achieved was similar to that with fresh tissue. 5. The results are discussed in relation to the previous report that the stimulation is observed throughout the neuronal cell-bodies and in relation to the hypothesis that the labelled phosphatidylinositol produced by stimulation is a component of an acetylcholine-receptor proteolipid localized in the synaptic junction.

Acetylcholine↗